Anal. Chem. 1998, 70, 2081-2084
An On-Line Electrophoretic Concentration Method for Capillary Electrophoresis of Proteins Xing-Zheng Wu,*,† Akihiko Hosaka,‡ and Toshiyuki Hobo‡
Department of Materials Science and Engineering, Faculty of Engineering, Fukui University, Bunkyo 3-9-1, Fukui-shi 910, Japan, and Department of Industrial Chemistry, Faculty of Engineering, Tokyo Metropolitan University, Minami Ohsawa, Hachioji, Tokyo 192-03, Japan
A simple on-line electrophoretic concentration method for capillary electrophoresis of proteins with a semipermeable hollow fiber is proposed. A short, semipermeable hollow fiber is connected to the inlet end of a capillary. An injection electric field is applied across the hollow fiber. Protein samples are electromigrated into the hollow fiber from a sample vial. Since the protein ions cannot pass through wall of the hollow fiber, protein ions are concentrated in the hollow fiber. After a certain period of injection (concentration) time, an electric field is applied across the hollow fiber and the capillary, and a conventional CE is carried out. The concentration effect of the method has been demonstrated successfully with model protein samples, cytochrome c, lysozyme, ribonuclease A, and r-chymotrypsinogen A. Experimental results show that the concentration factor is increased with the injection (concentration) time and the electrophoretic mobility of the proteins. When the injection (concentration) time is 60 s, CE detection limits of the model proteins can be lowered by 1000-fold for the present method. Factors affecting the concentration factors and the further improvements of the method are discussed. Capillary electrophoresis (CE) is growing as one of the most powerful analytical techniques with a wide range of applications because of its attractive features, such as high separation efficiency and short analysis time.1,2 In various detectors developed for CE, although mass sensitivity is extremely high because of the very small detection volumes in CE, concentration sensitivity is usually not enough. Particularly for the most widely used UV absorbance detection of CE, the concentration sensitivity is generally on the order of 10-100-fold less than that of HPLC due to the short optical path length in CE.3 So far, many efforts have been made to improve the concentration sensitivity in CE. There are basically two approaches, improving the capillary geometry and optical design, and sample concentration. Successful examples of the †
Fukui University. Tokyo Metropolitan University. (1) Li, S. F. Y. Capillary Electrophoresis: Principles, Practice, and Application; Elsevier: New York, 1992. (2) Kuhr, W. G.; Monnig, C. A. Anal. Chem. 1992, 64, 389R-407R. (3) Albin, M.; Grossman, P. D.; Moring, S. E. Anal. Chem. 1993, 65, 489A497A. ‡
S0003-2700(97)00911-6 CCC: $15.00 Published on Web 04/08/1998
© 1998 American Chemical Society
former involve use of a bent capillary (Z-cell),3,4 a silver-coated capillary (multireflection flow cell),5 end-column detection,6 offcolumn detection,7 and so on. There are also many reports on the sample concentration approach, including chemical concentration and electrophoretic concentration. The chromatographic concentration method, in which samples are first absorbed onto chromatographic packing materials,8 is a good example of the former. However, the manufacture of a reproducible on-line system seems to be a difficulty for this method. On the other hand, several successful on-line electrophoretic concentration methods have been reported. They are field amplification injection,9-11 stacking technique,11-13 and isotachophoresis (ITP).11,14,15. In these methods, discontinuous buffer systems are used in injection and separation processes. The concentration factors of these methods depend on the compositions of the discontinuous buffer systems. A good adjustment of the compositions of the discontinuous buffer systems is required in order to obtain a high concentration factor. Recently, Hjerten’s group has discussed concentration on a microliter scale of samples in detail, both theoretically and experimentally.16,17 Their results show that a concentration factor even higher than 1000-fold is possible by their three-step operations. More recently, they also proposed an interesting concentration method, making use of the transport of water by evaporation or the Donnan effect of a hollow fiber.18 In their experiments, diluted samples in a hollow fiber were concentrated into a narrow zone by removing water, and then the part of the hollow fiber (4) Chervet, J. P.; Van soest, R. E. J.; Ursem, M. J. Chromatogr. 1991, 543, 439-449. (5) Wang, T.; Aiken, J. H.; Huie, C. W.; Hartwick, R. A. Anal. Chem. 1991, 63, 1372-1376. (6) Xi, X.; Yeung, E. S. Appl. Spectrosc. 1991, 45, 1199-1203. (7) Wu, X.-Z.; Hosaka, A.; Kobayashi, E.; Hobo, T. J. Chromatogr. 1996, 726, 205-210. (8) Debets, A. J. J.; Mazereeuw, M.; Voogt, W. H.; van Iperen, D. J.; Lingeman, H.; Hupe, K.-P.; Brinkman, U. A. T. J. Chromatogr. 1992, 608, 151-158. (9) Burgi, D. S.; Chien, R.-L. Anal. Biochem. 1992, 202, 306-309. (10) Chien, R.-L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-496A. (11) Chien, R.-L.; Burgi, D. S. J. Chromatogr. 1991, 559, 153-161. (12) Gebauer, P.; Thormann, W.; Bocek, P. J. Chromatogr. 1992, 608, 47-57. (13) Aebersold, R.; Morrison, H. D. J. Chromatogr. 1990, 516, 79-88. (14) Stegehius, D. S.; Irth, H.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. 1992, 591, 341-349. (15) Foret, F.; Sustacek, V.; Bocek, P. J. Microcolumn Sep. 1990, 2, 229-233. (16) Hjerten, S.; Liao, J.-L.; Zhang, R. J. Chromatogr. 1994, 676, 409-420. (17) Liao, J.-L.; Zhang, R.; Hjerten, S. J. Chromatogr. 1994, 676, 421-430. (18) Zhang, R.; Hjerten, S. Anal. Chem. 1997, 69, 1585-1592.
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containing the concentrated zone was cut off. The concentrated zone was squeezed out and used as samples for CE. This method also provides a high concentration factor. However, to decrease loss in the handling of the minute sample volume, the number of steps or the operations should be minimized.18 Therefore, online concentration methods are more desirable for CE. In this work, we report an on-line electrophoretic concentration method for CE of proteins with a short, semipermeable hollow fiber. This method is different from other reported on-line electrophoretic concentration methods in that the same buffer solution is used in the injection and separation processes. Four basic proteins, cytochrome c, lysozyme, ribonuclease A, and chymotrypsinogen A, have been used as model protein samples. High concentration factors of the model proteins have been demonstrated.
EXPERIMENTAL SECTION Reagents and Sample Preparation. All solutions were prepared using deionized water and filtered with 3-mm pore size cellulose acetate filters. All chemicals were reagent grade and used as received. Protein samples were from Sigma, and other reagents were from Kantou Kagaku (Tokyo, Japan). A Tris (50 mM)-phorsphoric acid buffer solution (pH 3.0) was prepared by adding 1 M phosphoric acid solution into 50 mM Tris solution until the pH reached 3.0. The four model proteins were dissolved into the Tris-phosphoric acid buffer solution. Their concentration were 10-4 mg/mL, respectively. Capillary Electrophoresis Instrumentation. A high-voltage power supply (Glassman High Voltage) provided injection and separation voltages. An UV absorbance detector (CE-970, Japan Spectroscopic Co. Ltd.) was used for detection of the proteins; its wavelength was set as 215 nm. An untreated fused silica capillary with 50 µm i.d. and 360 µm o.d. (Polymicro Technologies) was used as a separation capillary. Its total length was 50 cm, and the length from the inlet end to the detector was 35 cm. The capillary was rinsed sequentially with 1 M NaOH, water, and the buffer solution for 30 min before use. Connection of a Short, Semipermeable Hollow Fiber to the Inlet End of the Capillary. First, one end of a hollow fiber (o.d., 200 µm; i.d., 150 µm; MWCO, 18 000; Spectrum Medical Industries, Inc., Los Angeles, CA) and the inlet end of the capillary were fixed in a Teflon tube (o.d., 1 mm; i.d., 0.5 mm; length, 5 cm) with epoxy resin (Figure 1). The length of the hollow fiber in the Teflon tube was kept as short as possible (about 2 mm). The capillary and hollow fiber were carefully arranged so that they were as coaxial as possible. Second, the capillary-Teflon tubehollow fiber was inserted into a V-shaped glass tube, and the end of the glass tube was sealed with epoxy resin (Figure 1). The length of the hollow fiber in the glass fiber was also kept as short as possible (about 2 mm). Injection (Concentration) and CE Procedure. The Vshaped glass tube was filled with the buffer solution, and a Pt electrode was inserted into it before experiments. In the sample injection (concentration) process, the V-shaped glass tube was inserted into a sample vial, and an injection voltage of 140 V was applied between the sample vial and the glass tube. As shown in Figure 1, the polarity of electrode in the glass tube was +, and that in the sample vial was -. After a certain period of injection 2082 Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
Figure 1. Illustration of experimental system for the on-line electrophoretic concentration method of CE.
(concentration), the injection voltage was off, and the sample vial was replaced by a buffer vial. Then, a separation voltage of 10 kV was applied across the two buffer vials, as shown by the dashed line in Figure 1, and CE of the protein samples was carried out. RESULTS AND DISCUSSIONS Since a semipermeable hollow fiber permits transportation of only small ions and molecules across their wall, it has been widely used in sampling19-21 and on-line desalting22 of CE. This characteristic of the hollow fiber is also used in the on-line electrophoretic concentration method. The four model proteins are in the form of cations in pH 3.0 Tris-phorsphoric acid buffer solution. As shown in Figure 1, when an injection voltage is applied between the sample vial and the V-shaped glass tube, cation ions in the sample vial will electroosmotically and electrophoretically migrate toward the V-shaped glass tube (for anion sample, polarity of electrodes in the sample vial and V-shaped glass tube are reversed). Small cations such as H+ can freely pass through the wall of the hollow fiber, while the protein cations cannot. Thus, the protein cations are stopped in the hollow fiber. This means that the protein cations are concentrated in the hollow fiber. The amount M (moles) of a protein concentrated into the hollow fiber for injection (concentration) time t (seconds) is calculated as follows:
M ) π(d/2)2UtC
(1)
where d (centimeters) is the inside diameter of the hollow fiber, and U (centimeters per second) and C (moles/milliliter) are electromigration velocity and concentration of the sample, respectively. Since the injection (concentration) electric field is applied locally at the hollow fiber and no electric field exists in the capillary in the injection (concentration) process, buffer solution in the capillary produces a great back pressure to electroosmotic flow in the hollow fiber.7 This means that the electroosmotic flow in the injection (concentration) process is negligible. Therefore, the electromigration velocity of the sample is equal to electrophoretic velocity Up (centimeters per second), which is proportional to the (19) Bao, L.; Dasgupta, P. K. Anal. Chem. 1992, 64, 991-996. (20) Kuban, P.; Karlberg, B. Anal. Chem. 1997, 69, 1169-1173. (21) Wu, X.-Z.; Wu, J.; Pawliszyn, J. Electrophoresis 1995, 16, 1474-1478. (22) Wu, J.; Pawliszyn, J. Anal. Chem. 1995, 67, 2010-2014.
Figure 3. Relations between peak height and the injection (concentration) time.
Figure 2. Electropherograms of the model proteins obtained with a conventional electromigration injection method (A) and the present method (B and C). The three electropherograms were of the same scale. In the conventional electromigration injection method, the injection electric field was 200 V/cm, and the injection time was 5 s. In the present on-line concentration method, the injection electric voltage was 140 V, and the injection (concentration) times were 30 (B) and 60 (C) s, respectively. Concentrations of the four proteins were all 10-4 mg/mL. Peaks 1-4 were cytochrome c, lysozyme, ribonuclease A, and R-chymotrypsinogen A, respectively.
injection electric field. Then, eq 1 is written as follows:
M ) π(d/2)2UptC
(2)
The volume V (milliliters) of the hollow fiber in the glass tube is
V ) π(d/2)2L
(3)
where L (centimeters) is the length of the hollow fiber in the V-shaped glass tube. The concentration C′ (moles/milliliter) of the protein sample in the hollow fiber calculated is as follows:
C′ ) M/V ) (1/L)UptC
(4)
The concentration factor C′/C is
C′/C ) (1/L)Upt
(5)
Equation 5 shows that the concentration factor is determined by the injection (concentration) time, the electrophoretic velocity of the sample, and the length of the hollow fiber. Figure 2 shows CE electropherograms of the model proteins for a conventional electromigration injection method and the present on-line electrophoretic concentration method. As shown in Figure 2A, the model proteins at a concentration level of 10-4 mg/mL could not be detected by the CE system when the samples were injected with a conventional electromigration method. However, the four proteins were easily detected with the on-line
electrophoretic concentration method. Figure 2B,C shows the electropherograms of the model samples for the on-line concentration method when the injection (concentration) times were 30 and 60 s, respectively. The peak height in Figure 2C was nearly 2 times that in Figure 2B. The experimental results of the relationship between the peak height and the injection (concentration) time are shown in Figure 3. The peak height (since a large error existed in the calculation of peak area, here the peak height instead of peak area is used) is considered to be proportional to the concentration factor. Therefore, Figure 3 shows that the concentration factor is proportional to the injection (concentration) time during 0-60 s. This is in accordance with the theoretical predication of eq 5. When the injection (concentration) time exceeded 60 s, peaks in the CE electropherogram seriously overlapped. This might be because the proteins were so concentrated that they exceeded the separation capability of the CE. Figure 3 also shows that the concentration factor was the largest for cytochrome c among the four proteins. This is also predicted by eq 5, since the electrophoretic velocity Up of cytochrome c was the largest among those of the four proteins in the experiments. Therefore, large concentration factors are obtained for protein ions with large electrophoretic mobilities. Since the electrophoretic mobility of a protein depends greatly on the pH of the buffer solution, the concentration factor is expected to be affected greatly by the pH of the buffer solution. The detection limits of the model proteins for a gravity flow injection method or an electrophoretic injection method were on the level of 10 -2 mg/mL for the CE systems. From S/N of Figure 2C, it is expected that the method could detect the model proteins even if their concentrations were 1 order lower, i.e., on the level of 10 -5 mg/mL, when the injection (concentration) time was 60 s. Therefore, the CE detection limit could be lowered by 1000fold by the present on-line concentration method. As stated below, it could be further improved if the connection between the hollow fiber and the capillary is improved or the injection (concentration) time is increased. As shown in eq 5, the concentration factor is proportional to 1/L. This means that the length of the hollow fiber in the glass tube should be near zero. The length of the hollow fiber also greatly affects the peak width in the electropherogram. The Analytical Chemistry, Vol. 70, No. 10, May 15, 1998
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shorter the length of the hollow fiber, the sharper the peak. Mismatch in the inner diameters of the hollow fiber and the capillary also contributed to broadening of the peaks. An ideal method for the on-line electrophoretic concentration technique might be use of a fracture7,23,24 at the inlet end of the capillary, over which a semipermeable membrane was coated. It should be pointed out that the model proteins used in this work are the most severely adsorbed ones on an untreated capillary wall.25,26 The adsorption of the proteins on the capillary wall also resulted
in broadening of peaks. Since the object of this work was to demonstrate the on-line concentration effect of the method, we did not treat the capillary. This method is not applicable to samples with small molecular weight since the hollow fiber is used. However, it is expected that this method was also useful for small ions if some selective membrane was used. The application of the method to other protein samples and small ions will be reported later.
(23) Linhares, M. C.; Kissinger, P. T. Anal. Chem. 1991, 63, 2076-2078. (24) Wu, X.-Z.; Sasaki, K.; Kobayashi, E.; Hobo, T. Bunseiki Kagaku 1994, 43, 609-613. (25) Liu, P. Z.; Malik, A.; Kuchar, M. C. J.; Lee, M. L. J. Microcolumn Sep. 1994, 6, 581-589. (26) Malik, A.; Zhao, Z.; Lee, M. L. J. Microcolumn Sep. 1993, 5, 119-125.
Received for review August 20, 1997. Accepted February 16, 1998.
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